ABSTRACT The overall goal of this project is to elucidate the impact of endocytic uptake mechanism and subsequent intracellular trafficking on the design criteria for polymeric gene delivery vectors. Efficient gene delivery requires vectors that are capable of navigating an intracellular obstacle course including endocytosis, escape from endocytic vesicles, transport through the cytoplasm to the nucleus, and nuclear import. For many therapies, vectors must first of all provide cell-specific targeting, most often through attachment of biological ligands that are recognized by receptors displayed on the desired cell type. Internalization of these receptors - and attached vectors - can occur via several processes that differ in the subsequent processing of the resulting vesicles. In particular, vectors internalized via clathrin-coated vesicles experience a rapid drop in pH and are trafficked to lysosomes whereas caveosomes are not acidified and avoid normal lysosomal degradation. In contrast, polymeric vectors, including proton-sponge materials such as polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimers, are often designed to provide escape from endocytic vesicles in response to vesicle acidification, without regard to internalization mechanism and vesicle processing. We hypothesize that endocytic processing of targeted gene delivery polymers depends on the mechanism by which they are internalized. In particular, clathrin and caveolin-mediated vector uptake require different design criteria for endocytic escape. In this project, a set of PEI and PAMAM gene delivery vectors have been designed that display either transferrin (Tf) or folate (FOL) for targeted uptake via clathrin- or caveolin-mediated pathways, respectively. The uptake mechanism of the various vectors in HeLa, MDA-MB-231 and SK-BR-3 cell lines will be confirmed quantitatively and qualitatively, and the effect of uptake mechanism on gene delivery will be determined (Aim 1). A series of established trafficking assays and biochemical inhibitors will be used to investigate the critical impact of uptake mechanism on vector acidification and endocytic vector escape (Aim 2). PEI derivatives with systematically varying buffering capacity will be synthesized to investigate the effects of varying response to vesicle acidification on endocytic escape and gene delivery activity (Aim 3). Finally, gene delivery efficiency of Tf- and FOL-targeted vectors will be evaluated in a sub-cutaneous murine tumor model (Aim 4). These experiments have been designed using well characterized polymers and targeting ligands to facilitate analysis and generalizability of the results. This project will result in new fundamental insight into structure-activity relationships of gene delivery polymers that will be useful to guide design of safe and efficient vectors for human gene therapy.